Flow cytometric sorting permits the selection, enrichment, apportionment, or division of populations of cells, viruses, bodies or particles of interest (hereinafter referred to as cells). The selection criteria include measurable properties of individual cells that can be detected from outside the cell, with or without the aid of chemical reagents or of complexes or bodies that are, or that may be caused to be, associated with the cell. For instance, properties of cells may be measured or approximated by detecting and/or quantifying the association of the cells with one or more labels, such as molecules, complexes, or bodies that fluoresce or have been modified to be rendered fluorescent. Such fluorescent molecules, complexes, and/or bodies may differentially associate with cells on the basis of qualitative or quantitative properties of the cells, including their composition with respect to proteins, lipids, phosphoproteins, glycoproteins, phospholipids, glycolipids, nucleic acids (including the quantity, sequence, or organization of nucleic acids), carbohydrates, salts/ions, and any other molecules in, on, or associated with the cells. Further, such fluorescent molecules, complexes, and/or bodies may differentially associate with cells based on physical or physiological characteristics of the cells, examples of which include but are not limited to membrane permeability, membrane composition, membrane fluidity, chemical or membrane potential, viability, chemical gradients, motility, reduction or oxidation potential or state, and other parameters or properties.
Other measurable properties of cells, whether labeled or unlabelled, modified or unmodified, that may provide a basis for cell selection may include but are not limited to:
properties of light interacting with the cells, such as fluorescence, absorbance, reflectance, scatter, polarization, or other properties;
electrical properties of the cells or of the effect of the cells on their environment, including conductance, inductance, resistance, membrane potential or voltage, or other properties;
magnetic or electromagnetic properties of cells, including magnetism, paramagnetism, magnetic resonance, and/or interaction of the cells with electromagnetic energy;
the appearance, image, or morphological properties of the cells; and
the makeup of the cells with respect to any substance or parameter, measured directly or indirectly in any way.
Furthermore, the measurement of such parameters, directly or indirectly, singularly or in combination, may reflect simple or complex properties of interest of the cells.
One example of such a property is the sex chromosome included in the diploid, haploid, or gamete genome, which may be an X chromosome or a Y chromosome or combinations of both depending on the cell type and the organism. The determination of sex chromosome content may be inferred using direct or indirect measurements or determinations using one or more methods. Such methods include the measurement of the DNA content of the cells determined relatively or absolutely; the presence or absence of certain DNA sequences, or markers of the presence or absence of certain DNA sequences; the size of the cells or of portions or organelles of the cells; the presence, localization, or absence of proteins or other markers characteristic of the sex chromosome content of the cells, or combinations or patterns of expression of such markers; or any other measurement that reflects the sex chromosome composition of the cell. Many other such measurements may be made, or properties determined, to identify cells that are of interest in a particular instance, situation, system, disease, condition, process, or circumstance.
Such cytometric measurements permit quantitative and/or qualitative determinations about cells, populations of cells, organs, tissues, or organisms. Such determinations may be used in many ways including but not limited to diagnosis, biomedical research, engineering, epidemiology, medicine, agriculture, animal husbandry, livestock management, zoology, biopharmaceutical industry, and other fields. In addition to the ability to perform such measurements, current methods and instrumentation permit the separation of cells based on characteristics or parameters measured by cytometry as described above. Cells can be selected positively or negatively by the concentration, collection, or partitioning of cells of interest or by the removal of cells that are not desired or of interest in the preparation. Such selection can be controlled on the basis of any parameter, characteristic, or combination of parameters or characteristics that can be determined as described above.
Cells identified by methods including or related to those described above can be separated, partitioned, concentrated, depleted, or collected into any arbitrary number of groups. One common separation method (depicted in FIG. 1A) uses electrostatic forces to divert an electrically or electrostatically charged stream, droplet, or droplets containing a cell or cells having desired or undesired properties. The diverted cells are collected or discarded as appropriate to the particular application, as illustrated in FIG. 1A. Other separation methods include the use of fluidic devices including valves to divert cells in a fluid stream to alternate pathways, channels, tubes, or elements for subsequent collection or disposal, as illustrated in FIG. 1B.
There exist a number of methods and systems for performing flow cytometric sorting of cells. Among these are methods and systems designed specifically to perform flow cytometric sorting of mammalian sperm cells and, in particular, to sort the sperm cells into populations of sperm cells bearing X chromosomes and/or populations of sperm cells bearing Y chromosomes, with the purpose of increasing the probability that fertilization of an egg with the sorted sperm will result in offspring with a desired gender. For example, a dairy farmer may desire to sort the sperm of a bull so that bovine embryos may be produced, by artificial insemination, in vitro fertilization, or other means, with sperm having an X chromosome to produce additional female bovine offspring.
Flow cytometric sorting methods present a number of challenges, particularly with respect to sorting mammalian sperm cells for later use in producing offspring. Importantly, methods used to label and/or to differentiate between the cells and/or methods used to sort the cells must not adversely affect the viability of the cells. Often, one or more goals of the methods and/or systems involved (e.g., faster sorting, improved accuracy, etc.) conflict with other goals of the methods and/or systems. Various factors must be balanced and considered, including the temperatures, temperature changes, pressures and/or pressure changes to which the cells are subjected, the fluidic environments to which the cells are exposed, the forces applied to the cells, and the lifespan of the cell. For example, the rate at which a fluorescent molecule (e.g., a fluorochrome) enters a cell to bind to DNA within the nucleus of the cell (i.e., the rate at which cells may be stained), may increase as temperature increases. Thus, the throughput of a system (at least the throughput of the staining process) may increase with an increase in the temperature of the cells' environment. However, increased temperature may prove detrimental to the viability of the cells and/or the length of time that the cells remain viable. By contrast, maintaining the cells at the optimal temperature for viability may increase the time required for staining (and measuring and sorting) the cells, such that the process takes longer than is practical or such that the cells are not viable after the time required to complete the process.
Another challenge associated with sorting cells relates to the physical and optical properties of the cells. In particular, flattened or otherwise asymmetrical cells, such as mammalian red blood cells or sperm cells, exhibit anisotropic emission of energy (e.g., light). The complex geometries of a cell's interior and/or the complex geometries of the cell's boundaries act to refract and/or reflect light in ways that are highly dependent on the orientation of the cell with respect to any illumination sources and/or detectors used to differentiate between cells. For example, flow cytometry sorting of mammalian sperm cells into populations having X or Y chromosomes usually involves staining the cells with a fluorescent molecule that binds to DNA within the cells. The variation in DNA content between the X and Y chromosomes of most mammalian species (Y chromosomes generally containing less DNA than X chromosomes) results in relatively greater fluorescence from cells containing X chromosomes. However, the difference in DNA content of X and Y chromosomes is typically on the order of only a few percent and, often, cell geometry and/or orientation can affect the detected fluorescence by a percentage that far exceeds the percentage difference in DNA content between the X and Y chromosomes. Additionally, such analysis requires that cells pass through the detection region singly, such that a detector does not interpret fluorescence from two cells as fluorescence from a single cell.
Flow cytometry sorting systems frequently employ a core-in-sheath fluidic mechanism to carry the cells through the detection region. As depicted in FIG. 1C, a relatively slow moving stream 50 of an aqueous suspension of cells 52 is injected into a relatively faster moving flow 54 of sheath fluid. This arrangement focuses the cells 52 into a stream 56, referred to as the core stream. With appropriate selection of the pressures and consequent velocities of the core suspension and sheath fluid, the core stream is narrowed by hydrodynamic forces exerted by the sheath flow, and the cells in the core stream are distributed longitudinally such that they are carried one by one in the flow. The forces that elongate and narrow the core stream have the additional benefit of orienting the cells 52 such that a lengthwise axis 58 of the cell 52 is parallel to the direction of flow of the single file stream 56. However, the orientation of the cells about the lengthwise axis 58 remains more or less random. Thus, as each cell 52 passes through the detection area, light incident upon the cell, light emitted from the cell (e.g., fluorescent light), and light reflected off of the cell, remains dependent on the orientation of the cell 52. This is especially true of many types of mammalian sperm cells.
There are a number of solutions to the problem of sperm cell orientation with respect to illumination and detection of cells within flow cytometry systems. For example, FIG. 1D illustrates one solution, which solution employs a cut, beveled tip 60 on a tube 62 injecting a sample stream 64 into a sheath flow 66. The flattened, beveled tip 60 helps to orient the cells about their lengthwise axes 58 within the sheath flow 66 such that the flat faces of the cells tend to align in a consistent direction. Another solution (which may be combined with the beveled tip solution) employs two detectors 68 and 70 orthogonal to each other (a 0-degree detector 68 and a 90-degree detector 70) which are used in combination to estimate the orientation of each cell 52 as it passes through a detection area 72 and to measure the fluorescence of those cells that are found to be appropriately oriented such that precise quantization of the fluorescent signal is possible. The solutions employing hydrodynamic orientation of cells around the lengthwise axis generally yield populations in which the desired alignment for fluorescence measurement is achieved for about 70% or less of the cells in the sample flow, which decreases the throughput of the instrument and results in the discarding of improperly oriented cells.
Still another solution to the problems associated with cell geometry and orientation utilizes optical detection along the same axis as the core-in-sheath flow that carries the cells. In one such solution, epi-illumination optics are used to illuminate the cell and detect light emitted by the cell. As depicted in FIG. 1E, a sample stream 74 carried by a sheath flow 76 travels directly towards a microscope objective lens 78, eliminating the dependence on the orientation of the cell (e.g., a sperm cell 80) about a lengthwise axis 82 of the cell 80. However, the trajectory of the cell 80 towards the objective lens 78 requires that the cell 80 change trajectory immediately after passing through a detection area 82 (i.e., the focal point 84 of the objective lens 78). The system accomplishes this trajectory change by using a transverse flow 86 of fluid. Uncertainty in the position of individual cells may be introduced after the analysis by the convergence 88 of the transverse fluid flow 86 and the sheath flow 76 and fluid stream 74. Such position uncertainty may render the system inoperable to perform cell sorting because the location of the cell 80 within the converged flow may become unpredictable immediately after the cell passes through the detection area 82.
Yet another solution, illustrated in FIG. 1F, utilizes one or more parabolic reflectors 102 to illuminate cells uniformly and/or to collect light radially from the cells. The system utilizes a nozzle 104 to emit a stream/jet 106 of liquid containing individual cells 92. The stream 106 moves through a detection region 94 and through a hole 96 in the parabolic reflector 102. At some point after passing through the detection region, the stream 106 is broken into droplets 90 which may be electrically charged. Thereafter, each of the droplets 90 may be sorted by, for example, deflecting the charged droplet 90 and electrically charged deflector plates 98 to deflect the droplets into one or more receptacles 100. Problematically, this “jet-in-air” configuration subjects the stream 106 (and the cells 92 contained within the stream 106) to a drop in pressure as the stream 106 exits the nozzle 104. Sudden changes in pressure (and the increased pressures within the nozzle itself), can adversely affect the viability of the cell 92 as can the subsequent impact of the cell 92 into the receptacle 100. Thus, the pressure and speed of the stream 106 exiting the nozzle 104 must remain below any threshold that could damage the cells 92, which decreases the throughput of the system. Additionally, the movement of the droplets 90 through the atmosphere may require environmental constraints including cleanliness of the room air (e.g., a “clean room”) and temperature-control.
Thus, even with the relatively advanced state of flow cytometry, there exists an ongoing need in the art to provide more efficient, more sensitive, and more precise methods of and devices for cell separation and/or identification.